Quantitative Evaluation of Noncovalent Interactions between 3,4-Dimethyl-1H-pyrazole and Dissolved Humic Substances by NMR Spectroscopy.

Nitrification inhibitors (NI) represent a valid chemical strategy to retard nitrogen oxidation in soil and limit nitrate leaching or nitrogen oxide emission. We hypothesized that humic substances can complex NI, thus affecting their activity, mobility, and persistence in soil. Therefore, we focused on 3,4-dimethylpyrazole phosphate (DMPP) by placing it in contact with increasing concentrations of model fulvic (FA) and humic (HA) acids. The complex formation was assessed through advanced and composite NMR techniques (chemical shift drift, line-broadening effect, relaxation times, saturation transfer difference (STD), and diffusion ordered spectroscopy (DOSY)). Our results showed that both humic substances interacted with DMPP, with HA exhibiting a significantly greater affinity than FA. STD emphasized the pivotal role of the aromatic signal, for HA-DMPP association, and both alkyl methyl groups, for FA-DMPP association. The fractions of complexed DMPP were determined on the basis of self-diffusion coefficients, which were then exploited to calculate both the humo-complex affinity constants and the free Gibbs energy (Kd and ΔG for HA were 0.5169 M and -1636 kJ mol-1, respectively). We concluded that DMPP-based NI efficiency may be altered by soil organic matter, characterized by a pronounced hydrophobic nature. This is relevant to improve nitrogen management and lower its environmental impact.

Hydrochar obtained with by-products from the sugarcane industry: Molecular features and effects of extracts on maize seed germination.

Sugarcane bagasse, vinasse and a mixture of sugarcane bagasse and vinasse were hydrothermally carbonized (HTC), with and without the addition of phosphoric acid, in order to propose new applications of sucroenergetic industry by-products on soil. Detailed information on the composition and properties of hydrochars has been obtained through elemental composition, thermogravimetric analysis, nuclear magnetic resonance and, thermochemolysis GC-MS. The soluble acidic fraction from the hydrochar samples were applied to maize seeds to evaluate the agronomic potential as biostimulants and relate the molecular features with maize seed germination. The HTC treatment converted polysaccharide-based biomasses into hydrochars with hydrophobic characteristics (C-Aryl and C-Akyl). Furthermore, the addition of phosphoric acid further increased the overall hydrophobicity and shifted the thermal degradation of the hydrochars to higher temperatures. Biomass influenced the hydrochars that formed, in which the molecular features of sugarcane bagasse determined the formation of more polar hydrochar, due to the preservation of lignin and phenolic components. Meanwhile, the HTC of vinasse resulted in a more hydrophobic product with an enrichment of condensed and recalcitrant organic fractions. The germination assay showed that polar structures of bagasse may play a role in improving the maize seeds germination rate (increase of ~11%), while the hydrophobic domains showed negative effects. The responses obtained in germination seems to be related to the molecular characteristics that organic extracts can present in solution.

Bio-Based Hydrogels Composed of Humic Matter and Pectins of Different Degree of Methyl-Esterification.

We prepared humo-pectic hydrogels through ionotropic gelation by crosslinking natural pectins of different degree of methyl-esterification with either humic substances (HS) extracted from cow manure compost or humic-like substances (HULIS) from depolymerized lignocellulose biorefinery waste. The hydrogels were characterized by solid-state 13C-NMR spectroscopy, scanning electron microscopy, spectroscopic magnetic resonance imaging and rheological analyses. Their ability to work as controlled release systems was tested by following the release kinetics of a previously incorporated model phenolic compound, like phloroglucinol. Our results indicated that the release properties of hydrogels were influenced by the molecular composition of HS and HULIS and by the different degrees of methyl-esterification of pectins. The hydrogel made by the high methoxyl pectin and HS showed the fastest rate of phloroglucinol release, and this was attributed not only to its morphological structure and crosslinking density but also to the least formation of ionic interactions between phloroglucinol and the polysaccharidic chains. Our study suggests that the efficiency of novel humo-pectic hydrogels as sustainable carriers of agroproducts to crops is related to a careful choice of the characteristics of their components.

Molecular characterization of organic matter in two calcareous soils: the effects of an acid decarbonation treatment.

The molecular composition of soil organic matter (SOM) of two calcareous soils highly rich in carbonates was assessed before and after decarbonation by acid washing with HCl through 13C-CPMAS-NMR spectroscopy and off-line thermochemolysis coupled with gas chromatography and mass spectrometry (THM-GC-MS). The acidic treatment promoted a considerable concentration of organic matter in both soils, thus improving the identification of molecules otherwise not easily detectable. Decarbonation induced only a slight loss of soil organic carbon (SOC), corresponding to 1.4 and 2.7% for A and B soils respectively. The acidic treatment also led to an increase in the organic carbon/total nitrogen (OC/N) ratio in soil A, while an opposite variation was found for the second soil. Moreover, variations in the concentration and molecular distribution of specific compound classes present in SOM were caused by the acid washing of soils. As confirmed by both 13C-CPMAS-NMR and thermochemolysis results, the molecules most susceptible to the acid treatment were the carbohydrates, lignin monomers (G14 and G15), fatty acids (C18 saturated and unsaturated), fatty acids of microbial origin (C15, C17, and C19), hydroxy acids (C16, C18), and dioic acids (C18) which represent the components weakly bound to the organic matrix. Our findings not only showed the efficacy of the decarbonation treatment of calcareous soils with 3 N HCl, but also indicated how the acidic washing can improve the differentiation of soils on the basis of SOM molecular characteristics. Graphical abstract.

Chlamyphilone, a Novel Pochonia chlamydosporia Metabolite with Insecticidal Activity.

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.

Isolation and Characterization of Gramineae and Fabaceae Soda Lignins.

Some agricultural residues such as wheat or barley straw, as well as certain fast-growing plants like Leucaena leucocephala and Chamaecytisus proliferus, could be used as raw materials for the paper industry as an alternative to traditional plants (eucalyptus, pine, etc.). In the present study, four types of lignin obtained from the spent liquors produced by the pulping processes using the abovementioned feedstocks were isolated and characterized. Lignin samples were acquired through an acid precipitation from these spent liquors. The characterization of the precipitated lignin samples were performed using a Fourier transform infrared spectroscopy (FT-IR) and both liquid- and solid-state nuclear magnetic resonance spectroscopy (NMR) to analyse the chemical structure, and thermogravimetric analysis (TGA) for determining the thermal properties. Additionally, chemical composition of lignin fractions was also measured. Even though they were of different botanical origin, all the studied samples except for wheat straw lignin had a similar chemical composition and thermal behaviour, and identical chemical structure. Wheat straw lignin showed a greater amount of Klason lignin and lower carbohydrate content. Furthermore, this lignin sample showed a higher thermal stability and significantly different cross-peak patterns in the 2D-NMR experiments. The molecular structures corresponding to p-coumarate (PCA), ferulate (FA) and cinnamyl aldehyde end-groups (J) were only detected in wheat isolated lignin.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

Interactions between natural organic matter and organic pollutants as revealed by NMR spectroscopy.

Natural organic matter (NOM) plays a critical role in regulating the transport and the fate of organic contaminants in the environment. NMR spectroscopy is a powerful technique for the investigation of the sorption and binding mechanisms between NOM and pollutants, as well as their mutual chemical transformations. Despite NMR relatively low sensibility but due to its wide versatility to investigating samples in the liquid, gel, and solid phases, NMR application to environmental NOM-pollutants relations enables the achievement of specific and complementary molecular information. This report is a brief outline of the potentialities of the different NMR techniques and pulse sequences to elucidate the interactions between NOM and organic pollutants, with and without their labeling with nuclei that enhance NMR sensitivity.

Acetone-induced polymerisation of 3-aminopropyltrimethoxysilane (APTMS) as revealed by NMR spectroscopy.

We followed the reactivity of acetone with 3-aminopropyltrimethoxysilane, a potential organosilane coupling agent, by (1)H, (13)C and (29)Si NMR spectroscopy. Selective 1D and 2D-edited NMR experiments significantly contributed to simplify the spectral complexity of reaction solution and elucidated molecular structures within progressive reaction phases. The course of the 3-aminopropyltrimethoxysilane reaction with acetone was shown by a progressive decrease of both reactants, and a concomitant appearance of water and methanol, due to formation of imine and hydrolysis of alkoxysilane groups, respectively. The occurrence of multiple siloxane linkages in a progressively larger cross-linked macromolecular structure was revealed by DOSY-NMR experiments and new signals in (29)Si-NMR spectra at different reaction times. The NMR approach described here may be applied to investigate the reactivity of other γ-aminopropylalkoxysilanes and contribute to define procedures for the preparation of silica-based materials.

A novel fungal metabolite with beneficial properties for agricultural applications.

Trichoderma are ubiquitous soil fungi that include species widely used as biocontrol agents in agriculture. Many isolates are known to secrete several secondary metabolites with different biological activities towards plants and other microbes. Harzianic acid (HA) is a T. harzianum metabolite able to promote plant growth and strongly bind iron. In this work, we isolated from the culture filtrate of a T. harzianum strain a new metabolite, named isoharzianic acid (iso-HA), a stereoisomer of HA. The structure and absolute configuration of this compound has been determined by spectroscopic methods, including UV-Vis, MS, 1D and 2D NMR analyses. In vitro applications of iso-HA inhibited the mycelium radial growth of Sclerotinia sclerotiorum and Rhizoctonia solani. Moreover, iso HA improved the germination of tomato seeds and induced disease resistance. HPLC-DAD experiments showed that the production of HA and iso HA was affected by the presence of plant tissue in the liquid medium. In particular, tomato tissue elicited the production of HA but negatively modulated the biosynthesis of its analogue iso-HA, suggesting that different forms of the same Trichoderma secondary metabolite have specific roles in the molecular mechanism regulating the Trichoderma plant interaction.

An Integrated Multilevel Approach Unveils Complex Seed-Nanoparticle Interactions and Their Implications for Seed Priming.

Nanotechnology has the potential to revolutionize agriculture with the introduction of engineered nanomaterials. However, their use is hindered by high cost, marginal knowledge of their interactions with plants, and unpredictable effects related to massive use in crop cultivation. Nanopriming is an innovative seed priming technology able to match economic, agronomic, and environmental needs in agriculture. The present study was focused on unveiling, by a multilevel integrated approach, undisclosed aspects of seed priming mediated by iron oxide magnetic nanoparticles in pepper seeds (Capsicum annuum), one of the most economically important crops worldwide. Inductively coupled plasma atomic emission mass spectrometry and scanning electron microscopy were used to quantify the MNP uptake and assess seed surface changes. Magnetic resonance imaging mapped the distribution of MNPs prevalently in the seed coat. The application of MNPs significantly enhanced the root and vegetative growth of pepper plants, whereas seed priming with equivalent Fe concentrations supplied as FeCl3 did not yield these positive effects. Finally, global gene expression by RNA-sequencing identified more than 2,200 differentially expressed genes, most of them involved in plant developmental processes and defense mechanisms. Collectively, these data provide evidence on the link between structural seed changes and an extensive transcriptional reprogramming, which boosts the plant growth and primes the embryo to cope with environmental challenges that might occur during the subsequent developmental and growth stages.

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture.

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.

Quantitative evaluation of noncovalent interactions between glyphosate and dissolved humic substances by NMR spectroscopy.

Interactions of glyphosate (N-phosphonomethylglycine) herbicide (GLY) with soluble fulvic acids (FAs) and humic acids (HAs) at pH 5.2 and 7 were studied by (1)H and (31)P NMR spectroscopy. Increasing concentrations of soluble humic matter determined broadening and chemical shift drifts of proton and phosphorus GLY signals, thereby indicating the occurrence of weak interactions between GLY and humic superstructures. Binding was larger for FAs and pH 5.2 than for HAs and pH 7, thus suggesting formation of hydrogen bonds between GLY carboxyl and phosphonate groups and protonated oxygen functions in humic matter. Changes in relaxation and correlation times of (1)H and (31)P signals and saturation transfer difference NMR experiments confirmed the noncovalent nature of GLY-humic interactions. Diffusion-ordered NMR spectra allowed calculation of the glyphosate fraction bound to humic superstructures and association constants (K(a)) and Gibbs free energies of transfer for GLY-humic complex formation at both pH values. These values showed that noncovalent interactions occurred most effectively with FAs and at pH 5.2. Our findings indicated that glyphosate may spontaneously and significantly bind to soluble humic matter by noncovalent interactions at slightly acidic pH and, thus, potentially pollute natural water bodies by moving through soil profiles in complexes with dissolved humus.

Cerinolactone, a hydroxy-lactone derivative from Trichoderma cerinum.

A novel metabolite, 3-hydroxy-5-(6-isopropyl-3-methylene-3,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)dihydrofuran-2-one, trivially named cerinolactone (1), has been isolated from culture filtrates of Trichoderma cerinum together with three known butenolides containing the 3,4-dialkylfuran-2(5H)-one nucleus, harzianolide (2), T39butenolide (3), and dehydroharzianolide (4). The structure of 1 was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests with the purified compound exhibited activity against Pythium ultimum, Rhizoctonia solani, and Botrytis cinerea.

Advanced printable hydrogels from pre-crosslinked alginate as a new tool in semi solid extrusion 3D printing process.

With the aim to overcome alginate shape fidelity issue during the semisolid extrusion 3D printing and matrix collapsing after drying, we speculated that a pre-crosslinking step of the alginate ink-gel with low amount of Ca+2 could improve the hydrogel performance. To verify this, the influence of pre-crosslinker concentration (10-25 mM) on the ink gel rheological properties were studied and flow behaviour and viscoelastic properties were determined. The developed ink gels were fully characterised by DSC and Magnetic Resonance Imaging (MRI). Moreover, extrudability and the shape retention of extruded forms after printing and after drying were studied. The rheological and MRI data, combined with the morphological analysis of printed forms allowed us to identify the relationship between printability, shape retention and shear thinning behaviour of gels, showing good extrudability for all the pre-crosslinked gels with a calcium concentration between 0.15 and 0.25, corresponding to both egg-box dimers and multimers interactions.

Carbon sequestration in soil by in situ catalyzed photo-oxidative polymerization of soil organic matter.

Here we describe an innovative mechanism for carbon sequestration in soil by in situ photopolymerization of soil organic matter under biomimetic catalysis. Three different Mediterranean soils were added with a synthetic water-soluble iron-porphyrin, irradiated by solar light, and subjected first to 5 days incubation and, then, 15, and 30 wetting and drying (w/d) cycles. The in situ catalyst-assisted photopolymerization of soil organic carbon (SOC) increased water stability of soil aggregates both after 5 days incubation and 15 w/d cycles, but not after 30 w/d cycles. Particle-size distribution of all treated soils confirmed the induced soil physical improvement, by showing a concomitant lower yield of the clay-sized fraction and larger yields of either coarse sand- or fine sand-size fractions, depending on soil texture, though only after 5 days incubation. The gain in soil physical quality was reflected by the shift of OC content from small to large soil aggregates, thereby suggesting that photopolymerization stabilized OC by both chemical and physical processes. A further evidence of the carbon sequestration capacity of the photocatalytic treatment was provided by the significant reduction of CO(2) respired by all soils after both incubation and w/d cycles. Our findings suggest that "green" catalytic technologies may potentially be the bases for future practices to increase soil carbon stabilization and mitigate CO(2) emissions from arable soils.

Metabolic profile of intact tissue from uterine leiomyomas using high-resolution magic-angle-spinning ¹H NMR spectroscopy.

High-resolution magic-angle-spinning (HRMAS) one- and two-dimensional (1)H and (13)C NMR spectroscopy was used to study intact healthy (myometrium) and benign (leiomyoma) uterine tissues of 10 patients. Twenty-eight metabolites were detected and assigned in both types of tissue. Principal component analysis (PCA) was applied to a conventional water-suppressed (1)H HRMAS NMR spectrum, and two NMR spectral editing methods, namely Carr-Purcell- Meiboom-Gill (CPMG) spin-echo and diffusion-edited techniques, were used. Only the PCA of CPMG spectra resulted in a good differentiation between the two tissue types. The CPMG spin-echo spectra were also useful in indicating depleted levels of taurine in uterine leiomyomas, which were well correlated with the histopathological determination. In addition, statistical analysis revealed that most leiomyomas contained elevated concentrations of glutamate and glutamine. Our results suggest that HRMAS represents a valuable adjunct to histopathology to improve the diagnostic accuracy of uterine leiomyomas, whilst concomitantly reducing the diagnosis time.

The fate of cigarette butts in different environments: Decay rate, chemical changes and ecotoxicity revealed by a 5-years decomposition experiment.

Cigarette butts (CBs) are the most common litter item on Earth but no long-term studies evaluate their fate and ecological effects. Here, the role of nitrogen (N) availability and microbiome composition on CBs decomposition were investigated by a 5-years experiment carried out without soil, in park grassland and sand dune. During decomposition, CBs chemical changes was assessed by both 13C CPMAS NMR and LC-MS, physical structure by scanning electron microscope and ecotoxicity by Aliivibrio fischeri and Raphidocelis subcapitata. Microbiota was investigated by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. CBs followed a three-step decomposition process: at the early stage (∼30 days) CBs lost ∼15.2% of their mass. During the subsequent two years CBs decomposed very slowly, taking thereafter different trajectories depending on N availability and microbiome composition. Without soil CBs showed minor chemical and morphological changes. Over grassland soil a consistent N transfer occurs that, after de-acetylation, promote CBs transformation into an amorphous material rich in aliphatic compounds. In sand dune we found a rich fungal microbiota able to decompose CBs, even before the occurrence of de-acetylation. CBs ecotoxicity was highest immediately after smoking. However, for R. subcapitata toxicity remained high after two and five years of decomposition.

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

HRMAS-NMR metabolomics of Aglianicone grapes pulp to evaluate terroir and vintage effects, and, as assessed by the electromagnetic induction (EMI) technique, spatial variability of vineyard soils.

Aglianicone is an autochthonous variety of black berried wine grape cultivated exclusively in southern Italy, but with limited information on the molecular composition of its grapes. HRMAS NMR spectroscopy was used to identify the primary metabolome of Aglianicone grape pulp from two different vineyards and the observed metabolomic changes reflected the local terroir conditions or harvesting years. Moreover, the berries pulp metabolome was related to Apparent Electrical Conductivity (ECa) maps deriving from on-site near-surface geophysical measurements of vineyard soils obtained by an electromagnetic induction (EMI) technique. Most of primary metabolites of Aglianicone grape varied systematically as a function of the spatial variability of soil properties as well as vineyards terroir and harvesting years. Therefore, the proposed approach that couples HRMAS NMR spectroscopy with ECa maps of vineyard soils represents an innovative support for wine producers who can select the best vine/soil combination to ensure the maximum wine quality.